Cloning and Sequencing of the Ferulate Esterase Gene From Lactobacillus Buchneri LN4017

ABSTRACT

Embodiments of the present invention include polypeptides having ferulate esterase activity and the nucleic acid sequences that encode them. Methods of the embodiments utilize these ferulate esterase polypeptides and nucleic acid sequences to enhance the digestibility of plant cell walls and the accessibility of carbohydrates in plants. The invention provides for transgenic plants and bacteria transformed with expression vectors containing a DNA sequence encoding ferulate esterase from  Lactobacillus buchneri . Methods of using same to enhance plant fiber digestion in animals, are disclosed. Uses of this invention include, but are not limited to, forage and silage with improved digestibility for livestock, and enhanced biomass conversion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/939,343 filed Nov. 13, 2007, which claims the benefit of U.S.Provisional Application No. 60/865,492 filed Nov. 13, 2006.

FIELD OF THE INVENTION

The present invention relates to polypeptides having ferulate esteraseactivity and the nucleic acid sequences that encode them, and methods ofuse to enhance cell wall digestibility in plants.

BACKGROUND OF THE INVENTION

The plant cell wall is a complex structure consisting of differentpolysaccharides, the major components being cellulose, hemicellulosesand pectins. These polysaccharides may be cross-linked, or linked tolignin by phenolic acid groups such as ferulic acid. Ferulic acid mayplay a role in the control of cell wall growth in the plant and ferulicacid cross-linking within the cell wall is believed to restrict cellwall digestion by microorganisms (Fry et al., (1983) Planta 157:111-123; and Borneman et al., (1990) Appl. Microbial. Biotechnol. 33:345-351). The resistance of the plant cell wall to digestion presentssignificant challenges in the animal production industry. Somemicroorganisms are known to exhibit ferulic acid esterase activity(ferulate esterase) and thereby facilitate the breakdown of plant cellwalls and fiber digestion (U.S. Pat. No. 6,143,543).

Plant cell walls contain a range of alkali-labile ester-linked phenolicacids. In particular, grass cell walls are characterized by the presenceof large amounts of esterified ferulic and p-coumaric acids (mainly intheir E configurations), linked to arabinoxylans at the C5 of arabinose.These are released as ferulated oligosaccharides (FAX and PAX) bycellulase treatment but in vivo provide a substrate forperoxidase-catalyzed cross-linking of cell wall polysaccharides andlignin. The high levels of these phenolic acids and their dimers have adramatic influence on the mechanical properties, digestibility and ratesof digestion of grasses by ruminants.

It has been shown that ferulic acid is the predominant p-hydroxycinnamicacid esterified to grass polysaccharide. Dehydrodiferulate dimers andcyclobutane-type dimer mixtures have been isolated from plant cell walls(Waldron, et al., (1996) Phytochemical Analysis 7:305). These mixturesare present in large amounts in grass cells. Ether linked ferulicacid-coniferyl alcohol dimers, have also been isolated from cell walls(Jacquet, et al., (1996) Polyphenol Comm. Bordeaux pp 451) establishingthat ferulate esters are oxidatively co-polymerized with ligninprecursors which may anchor lignins to cell wall polysaccharides. Theyield of these dimers in grass cells indicates that phenolicdehydrodimer cross-linking of cell wall polysaccharides is much moreextensive than was previously thought.

An enzyme system has been reported from parsley endomembranes thatcatalyses the ferulation of endogenous polysaccharide acceptors fromferuloyl CoA, pointing to the ER/golgi as the site of polysaccharideesterification and the CoA ester as the physiological co-substrate(Meyer, et al., (1991) FEBS Lett. 290:209). Further evidence for thishas been found in water-soluble extracellular polysaccharides excretedin large amounts into the medium by grass cell cultures. This materialis highly esterified with ferulic and p-coumaric acid at levels similarto the cell walls of the cultured cells.

Ferulate esterase activity has been detected in several fungal speciesincluding, anaerobic gut fungi, yeasts, actinomycetes, and a fewfiber-degrading ruminal bacteria, which enables them to de-esterifyarabinoxylans and pectins.

Presently in livestock agriculture, while a high-forage diet isdesirable, it does not currently satisfy the demands of modern animalproduction. Fiber digestion is a limiting factor to dairy herd milkyield and composition, and to beef production in beef operations feedinga high forage diet, and hence restricts profitability of farmers.Enhancing fiber digestion has a dual impact: 1) the animal eats more dueto a reduced gut fill and therefore produces more, and 2) the animalgets more out of what it eats since the fiber is more digestible.Ultimately, these changes should increase milk yield, in dairy cows, andbeef production in forage fed animals. Farmers either have to choosewhether to tolerate a lower level of feed digestibility and henceproductivity, or they can choose to use inoculants, forage additives orother amendments that improve the digestibility of feed.

Accordingly, farmers can treat ensiled feed or other animal feed withfiber degrading enzymes, originating mainly from molds, to improvedigestibility of feed. In addition, there are several commerciallyavailable Saccharomyces cerevisiae yeast strains that when fed to cattlereportedly improve fiber digestion (Erasmus et al., (1992) J. Dairy Sci.75: 3056-3065; and Wohlt et al., (1998) J. Dairy Sci. 81: 1345-1352).Another alternative approach to improving fiber digestion is theprovision of a diet inherently possessing good digestibilitycharacteristics. For corn silage, this may include brown midrib cornsilage (Oba and Allen, (1999) J. Dairy Sci. 82: 135-142), oralternatively, corn hybrids recognized as being highly digestible.Further, new technologies incorporate fungal gene(s) responsible for theproduction of ferulate esterase into plant tissue for subsequentexpression, resulting in improvements in fiber digestibility (WO02/68666).

Generally, for an animal to make efficient use of the feed it consumes,the energy demands of the microorganisms in the digestive tract must bemet and synchronized with the availability of plant proteins. A lack ofsynchrony will lead to a) proteins and other nutrients being poorlyutilized in the digestive tract, b) a loss of nitrogen, in urine andfeces and c) a need to feed excessive amounts of protein concentrates assupplements to the diet. The use of organisms and enzymes can improve orenhance the value of the feed animals receive and the performance of theanimals. For example, WO 92/10945 discloses such a combination for usein enhancing the value of prepared silage. WO 93/13786 and WO 96/17525relate to the enhancement of animal performance using microorganisms,while WO 93/3786 refers to a species of Lactobacillus. Further, it hasbeen shown that Lactobacillus buchneri is suitable as a direct fedmicrobial to increase an animal's performance (U.S. Pat. No. 6,699,514).

BRIEF SUMMARY OF THE INVENTION

Provided herein are compositions and methods for improving feeddigestibility. One embodiment provides a DNA construct comprising apromoter that drives expression in a plant or plant cell, operablylinked to a nucleotide sequence that encodes a polypeptide havingferulate esterase activity, wherein the nucleotide sequence is apolynucleotide comprising SEQ ID NO: 1, 2 or 4, or a polynucleotide withat least about 85% sequence identity to SEQ ID NO: 1, 2 or 4, such as apolynucleotide encoding SEQ ID NO: 3 or 5, or a polynucleotide encodinga polypeptide having at least 85% sequence identity to SEQ ID NO: 3 or5. The nucleotide sequence of the construct may be derived fromLactobacillus buchneri or from other microbial sources, or it may be anengineered sequence that has been optimized, shuffled, or otherwisesubjected to engineered variation. The DNA construct may or may notinclude signal peptides or targeting sequences.

Another embodiment is a transformed plant or plant cell comprising theDNA construct described above. Such plants or plant cells may be monocotplants or cells, or dicot plants or cells. Particularly, such plants orplant cells may include plants or cells from Festuca, Lolium, Sorghum,Zea, Triticum, Avena, and Poa. Such plants may display increaseddigestibility. Seeds of these plants that comprise the DNA construct arealso embodiments of the invention. Additional embodiments include suchtransgenic plants as described above which also comprise an introducedDNA sequence encoding a xylanase.

Further embodiments include a method of controlling the level ofphenolic acid cross-linking in plant cell walls of a transgenic plant,the method comprising introducing into the plant a DNA constructcomprising a promoter that drives expression in a plant or plant cell,operably linked to a nucleotide sequence that encodes a polypeptidehaving ferulate esterase activity, wherein the nucleotide sequence is apolynucleotide comprising SEQ ID NO: 1, 2 or 4, or a polynucleotide withat least about 85% sequence identity to SEQ ID NO: 1, 2 or 4, such as apolynucleotide encoding SEQ ID NO: 3 or 5, or a polynucleotide encodinga polypeptide having at least 85% sequence identity to SEQ ID NO: 3 or5. Transgenic plants, plant parts and seeds produced by such a methodare also encompassed by the embodiments.

Another embodiment is a method for increasing digestibility of a plantor plant part fed to an animal, the method comprising introducing into aplant a DNA construct comprising a promoter that drives expression in aplant or plant cell, operably linked to a nucleotide sequence thatencodes a polypeptide having ferulate esterase activity, wherein thenucleotide sequence is a polynucleotide comprising SEQ ID NO: 1, 2 or 4,or a polynucleotide with at least about 85% sequence identity to SEQ IDNO: 1, 2 or 4, such as a polynucleotide encoding SEQ ID NO: 3 or 5, or apolynucleotide encoding a polypeptide having at least 85% sequenceidentity to SEQ ID NO: 3 or 5. Transgenic plants, plant parts and seedsproduced by such a method are also encompassed by the embodiments.

The embodiments also include isolated polypeptides comprising the aminoacid sequence set forth in SEQ ID NOs: 3 or 5; or a polypeptide havingat least 85% sequence identity to SEQ ID NOs: 3 or 5, wherein thepolypeptide has ferulate esterase activity. Furthermore, the embodimentsinclude isolated nucleic acid molecules, including a polynucleotidecomprising the sequence set forth in SEQ ID NOs: 1, 2 or 4; apolynucleotide having at least about 85% sequence identity to SEQ IDNOs: 1, 2 or 4; a polynucleotide encoding the amino acid sequence of SEQID NOs: 3 or 5; and a polynucleotide encoding the amino acid sequence ofa polypeptide having at least 85% sequence identity to SEQ ID NOs: 3 or5, wherein said polypeptide has ferulate esterase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: GAP alignment of SEQ ID NO: 3 with CinII (SEQ ID NO: 7) fromButyrivibrio fibrisolvens (AAB57776.1) showing conservation of thecatalytic triad.

FIG. 2: GAP alignment of SEQ ID NO: 3 with Cin I (SEQ ID NO: 6) fromButyrivibrio fibrisolvens (AAC44493.1) showing conservation of thecatalytic triad.

FIG. 3: Map of the region sub-cloned for sequence analysis. Deletionswere constructed by using convenient restriction enzymes to digest andre-ligate or to generate fragments for sub-cloning. The direction oftranscription by the plasmid borne lac promoter is indicated for eachplasmid. The direction of translation of open reading frames (ORFS)identified from the DNA sequence is indicated by arrows. Restrictionenzyme sites are shown as follows: H, HindIII; E, EcoRI.

FIG. 4: GAP alignment of SEQ ID NO: 3 with SEQ ID NO: 5 showingconservation of the catalytic triad.

FIG. 5: GAP alignment of SEQ ID NO: 5 with Cin I (SEQ ID NO: 6) fromButyrivibrio fibrisolvens (AAC44493.1) showing conservation of thecatalytic triad.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods directed toproducing ferulate esterase (FE) and acetyl esterase (AE) and using saidFE to enhance silage plant fiber digestion in animals. The compositionsare nucleotide and amino acid sequences for FE enzymes. Those of skillin the art are aware that FE is known by a number of different names,including, but not limited to, feruloyl esterase, cinnamoyl esterhydrolase, ferulic acid hydrolase, and hydroxycinnamoyl esterase.Changes in the naming of enzymes can occur frequently, so theInternational Union of Biochemistry and Molecular Biology (IUBMB) has anomenclature system to standardize the names of enzymes by assigningnumbers to them. The IUBMB number for the ferulate esterase (FE)referred to throughout this disclosure is EC 3.1.1.73. The IUBMB numberfor the acetyl esterase (AE) referred to throughout this disclosure isEC 3.1.3.6. Specifically, the present invention provides FE polypeptideshaving the amino acid sequences set forth in SEQ ID NOs:3 and 5, andvariants and fragments thereof. Nucleic acid molecules that wereisolated from the strain of Lactobacillus buchneri designated LN4017,such as, for example, SEQ ID NO:1, and variants and fragments thereof,comprising nucleotide sequences that encode the amino acid sequencesshown in SEQ ID NOs: 3 and 5 are further provided.

A deposit of the Lactobacillus buchneri LN4017 strain was previouslymade with the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209 (ATCC Accession No. PTA-6138). Theseorganisms were deposited on Aug. 3, 2004, as required for U.S. patentapplication Ser. No. 11/217,764, herein incorporated by reference. Themicroorganisms deposited with the ATCC were taken from the same depositmaintained at Pioneer Hi-Bred International, Inc (Des Moines, Iowa).Applicant(s) will meet all the requirements of 37 C.F.R. §1.801-1.809,including providing an indication of the viability of the sample whenthe deposit is made. Each deposit will be maintained without restrictionin the ATCC Depository, which is a public depository, for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced if it everbecomes nonviable during that period. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernment action.

Nucleotide sequences that are optimized for expression in plants, andthat encode the polypeptides of SEQ ID NOs: 3 and 5 can be generatedusing standard methods known in the art. Such plant-optimized nucleotidesequences are further encompassed by the present invention. Plants,plant cells, seeds, and microorganisms comprising a nucleotide sequencethat encodes an FE polypeptide of the invention are also disclosedherein. Compositions comprising an isolated FE polypeptide or amicroorganism that expresses a polypeptide of the invention are furtherprovided. The compositions of the invention find use in methods ofchanging the cell wall structure of transgenic plants and therefore,making them more digestible. The method comprises introducing an FEcoding sequence into the cells of a plant. Operably linked to the codingsequence is a promoter that can be either constitutive or inducible andsignal sequences that serve to target expression of the coding sequencein the desired organelle in the desired cell of the plant. The signalsequences can be either or both N terminal or C terminal sequences.Optionally, a second and/or third coding sequence is introduced into theplant, such as a xylanase coding sequence, which may be co-expressedwith the FE coding sequence.

“Xylanase” refers to a well described class of glycosyl hydrolases thathydrolyze xylan. Commercial applications of xylanase include thedegradation and bleaching of wood pulp for paper making. Xylanase canalso be added to animal feed to improve the digestibility of plantmatter. Typically, commercial xylanase is derived from fungi, such asTrichoderma.

The FE polypeptide SEQ ID NO:3 shares homology with some previouslyknown alpha/beta hydrolases, Lactobacillus esterases and cinnamoyl esterhydrolases. Members of the alpha/beta hydrolase-fold family of enzymescontain a catalytic triad of amino acids consisting ofnucleophile-acid-histidine. These three amino acids are separated by avariable number of amino acid residues in the primary sequence, but arelocated in similar topological locations in the native protein. In mostcases, the nucleophile is a serine residue. (Diaz, E., Timmis, K. N.(1995) The Journal of Biological Chemistry Vol. 270 No. 11, 6403-6411).Dalrymple proposed active site residues for cinI and cinII (Dalrymple,B. P. & Swadling, Y. (1997). Microbiology 143, 1203-1210. and Dalrymple,B. P., Swadling, Y., Cybinski, D. H. & Xue, G. P. (1996) FEMS MicrobiolLett 143, 115-120). BlastX analysis of ORF1 from SEQ ID NO:1 (which isset forth separately in SEQ ID NO: 2) demonstrated homology to CinII andCinI with conservation of the proposed catalytic triad (Ser, Asp andHis) of Cin II and CinI within the query sequence (See GAP alignmentsset forth in FIGS. 1 and 2).

To determine the presence of or an increase of FE activity, an enzymaticassay can be used. These assays are readily available in the literatureand those of skill in the art can readily find them. One of skill willrecognize that other assays can be used to detect the presence orabsence of FE. These assays include but are not limited to; immunoassaysand electrophoretic detection assays (either with staining or westernblotting). See, for example, Huggins and Lapides (1947) J. Biol. Chem.170: 467-482; and Mastihuba et al. (2002) Analytical Biochemistry 309,96-101.

In particular aspects, embodiments of the invention provide for methodsof changing the cell wall structure of transgenic plants and therefore,making them more digestible. The method comprises introducing a ferulicacid esterase coding sequence into the cells of a plant, operably linkedto a promoter that drives expression in the plant. The plant expressesthe FE polypeptide, thereby causing newly conferred or increased FEexpression in the plant. Expression of an FE polypeptide of theinvention may be targeted to specific plant tissues where FE isparticularly important, such as, for example, the leaves, stalks, orvascular tissues. Such tissue-preferred expression may be accomplishedby tissue-preferred promoters, such as leaf-preferred, vasculartissue-preferred, or stalk-preferred promoters. Similarly, the timing ofFE expression may be of significant importance, and accordingly,temporal promoters may be desired. For example, if expression of FE isdesired after the plant has been cut, removed from the ground oringested, an appropriate promoter would be a senescence promoter. Forexample, the promoter of BFN1 could be used, since BFN1 has recentlybeen shown to be a nuclease expressed in senescing leaves, Perez-Amador,et al., (2000) Plant Physiol. 122:169. Similarly, the promoter of SAG12,a cysteine protease which is also found in senescing leaves, could beused (Noh & Amasino, (1999) Plant Mol. Biol. 41:181). Moreover, thepolypeptides of the invention may also be targeted to specificsubcellular locations within a plant cell.

Optionally, a second and/or third coding sequence is introduced into theplant, such as, for example, a xylanase coding sequence, which could beco-expressed with the FE coding sequence.

Just as expression of an FE polypeptide of the invention may be targetedto specific plant tissues or cell types through the use of appropriatepromoters, it may also be targeted to different locations within thecell through the use of targeting information or “targeting labels.”Unlike the promoter, which acts at the transcriptional level, suchtargeting information is part of the initial translation product.Depending on the metabolic function of the tissue or cell type, thelocation of the protein in different compartments of the cell may makeit more efficacious or make it interfere less with the functions of thecell. For example, one may produce a protein preceded by a signalpeptide, which directs the translation product into the endoplasmicreticulum, by including in the construct (i.e. expression cassette)sequences encoding a signal peptide (such sequences may also be calledthe “signal sequence”). The signal sequence used could be, for example,one associated with the gene encoding the polypeptide, or it may betaken from another gene.

There are many signal peptides described in the literature, and they arelargely interchangeable (Raikhel and Chrispeels, “Protein sorting andvesicle traffic” in Buchanan et al., eds, (2000) Biochemistry andMolecular Biology of Plants (American Society of Plant Physiologists,Rockville, Md.), herein incorporated by reference). The addition of asignal peptide will result in the translation product entering theendoplasmic reticulum (in the process of which the signal peptide itselfis removed from the polypeptide), but the final intracellular locationof the protein depends on other factors, which may be manipulated toresult in localization most appropriate for the polypeptide and celltype. The default pathway, that is, the pathway taken by the polypeptideif no other targeting labels are included, results in secretion of thepolypeptide across the cell membrane (Raikhel and Chrispeels, supra)into the apoplast. The apoplast is the region outside the plasmamembrane system and includes cell walls, intercellular spaces, and thexylem vessels that form a continuous, permeable system through whichwater and solutes may move. This will often be a suitable location.

Alternatively, the use of vacuolar targeting labels such as thosedescribed by Raikhel and Chrispeels, supra, in addition to a signalpeptide will result in localization of the peptide in a vacuolarstructure. As described in Raikhel and Chrispeels, supra, the vacuolartargeting label may be placed in different positions in the construct.Use of a plastid transit peptide encoding sequence instead of a signalpeptide encoding sequence will result in localization of the polypeptidein the plastid of the cell type chosen (Raikhel and Chrispeels, supra).Such transit peptides are known in the art. See, for example, Von Heijneet al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J.Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481. Chloroplasttargeting sequences that encode such transit peptides are also known inthe art and include the chloroplast small subunit ofribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filhoet al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J.Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphatesynthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb.22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem.270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem.272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J.Biol. Chem. 268(36):27447-27457); and the light harvesting chlorophylla/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem.263:14996-14999). A person skilled in the art could also envisiongenerating transgenic plants in which the chloroplasts have beentransformed to over-express a gene for an FE peptide. See, for example,Daniell (1999) Nature Biotech 17:855-856; and U.S. Pat. No. 6,338,168.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. Polypeptides of the invention can be produced either from anucleic acid disclosed herein, or by the use of standard molecularbiology techniques. For example, a truncated protein of the inventioncan be produced by expression of a recombinant nucleic acid of theinvention in an appropriate host cell, or alternatively by a combinationof ex vivo procedures, such as protease digestion and purification.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.“Fragment” is intended to mean a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence have FEactivity. Alternatively, fragments of a nucleotide sequence that areuseful as hybridization probes generally do not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequencemay range from at least about 20 nucleotides, about 50 nucleotides,about 100 nucleotides, and up to the full-length nucleotide sequenceencoding the polypeptides of the invention.

A fragment of a nucleotide sequence that encodes a biologically activeportion of a polypeptide of the invention will encode at least 15, 25,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 245, 250, or 255 contiguous amino acids,or up to the total number of amino acids present in a full-lengthpolypeptide of the invention (for example, 260 amino acids for SEQ IDNO:3). Fragments of a nucleotide sequence that are useful ashybridization probes or PCR primers generally need not encode abiologically active portion of an FE protein.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide means having the entire nucleic acid sequence of a nativesequence. “Native sequence” is intended to mean an endogenous sequence,i.e., a non-engineered sequence found in an organism's genome.

Thus, a fragment of a nucleotide sequence of the invention may encode abiologically active portion of an FE polypeptide, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of an FEpolypeptide can be prepared by isolating a portion of one of thenucleotide sequences of the invention, expressing the encoded portion ofthe FE protein (e.g., by recombinant expression in vitro), and assessingthe activity of the encoded portion of the FE protein. Nucleic acidmolecules that are fragments of a nucleotide sequence of the inventioncomprise at least 15, 20, 50, 75, 100, or 150 contiguous nucleotides, orup to the number of nucleotides present in a full-length nucleotidesequence disclosed herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. One of skill in the artwill recognize that variants of the nucleic acids of the invention willbe constructed such that the open reading frame is maintained. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the FE polypeptides of the invention. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotide, such as those generated, for example, by usingsite-directed mutagenesis but which still encode an FE protein of theinvention. Generally, variants of a particular polynucleotide of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 3 is disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, FE activity as described herein. Such variantsmay result from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native FE protein of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a protein of theinvention may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the FE proteinsof the embodiments can be prepared by mutations in the DNA. Methods formutagenesis and polynucleotide alterations are well known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein. Guidance as to appropriate amino acid substitutions thatdo not affect biological activity of the protein of interest may befound in the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal. Such individual substitutions, deletions or additions to apolypeptide or protein sequence which alter, add or delete single aminoacids or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant.” Such an alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,        Creighton, Proteins (1984)).

Thus, the genes and polynucleotides of the invention include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired FE activity. Obviously, the mutations that will bemade in the DNA encoding the variant must not place the sequence out ofreading frame and optimally will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent No. 0075444.

In nature, some polypeptides are produced as complex precursors which,in addition to targeting labels such as the signal peptides discussedelsewhere in this application, also contain other fragments of peptideswhich are removed (processed) at some point during protein maturation,resulting in a mature form of the polypeptide that is different from theprimary translation product (aside from the removal of the signalpeptide). “Mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor protein” or“prepropeptide” or “preproprotein” all refer to the primary product oftranslation of mRNA; i.e., with pre- and propeptides still present. Pre-and propeptides may include, but are not limited to, intracellularlocalization signals. “Pre” in this nomenclature generally refers to thesignal peptide. The form of the translation product with only the signalpeptide removed but no further processing yet is called a “propeptide”or “proprotein.” The fragments or segments to be removed may themselvesalso be referred to as “propeptides.” A proprotein or propeptide thushas had the signal peptide removed, but contains propeptides (herereferring to propeptide segments) and the portions that will make up themature protein. The skilled artisan is able to determine, depending onthe species in which the proteins are being expressed and the desiredintracellular location, if higher expression levels might be obtained byusing a gene construct encoding just the mature form of the protein, themature form with a signal peptide, or the proprotein (i.e., a formincluding propeptides) with a signal peptide. For optimal expression inplants or fungi, the pre- and propeptide sequences may be needed. Thepropeptide segments may play a role in aiding correct peptide folding.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by assays that measure FE activity (See, for example, Hugginsand Lapides (1947) supra, Mastihuba et al. (2002) supra.).

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different FE proteincoding sequences can be manipulated to create a new FE proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the FE proteingene of the invention and other known FE protein genes to obtain a newgene coding for a protein with an improved property of interest, such asincreased FE activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The polynucleotides of the invention can be used to isolatecorresponding sequences from other organisms, particularly othermicroorganisms, more particularly other bacteria. In this manner,methods such as PCR, hybridization, and the like can be used to identifysuch sequences based on their sequence homology to the sequences setforth herein. Sequences isolated based on their sequence identity to theentire sequences set forth herein or to variants and fragments thereofare encompassed by the present invention. Such sequences includesequences that are orthologs of the disclosed sequences. “Orthologs” isintended to mean genes derived from a common ancestral gene and whichare found in different species as a result of speciation. Genes found indifferent species are considered orthologs when their nucleotidesequences and/or their encoded protein sequences share at least 60%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orgreater sequence identity. Functions of orthologs are often highlyconserved among species. Thus, isolated polynucleotides that encode foran FE protein and which hybridize under stringent conditions to thesequences disclosed herein, or to variants or fragments thereof, areencompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the polynucleotides of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989) supra.

For example, an entire polynucleotide disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding polynucleotides and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among FE polynucleotidesequences and are optimally at least about 10 nucleotides in length, andmost optimally at least about 20 nucleotides in length. Such probes maybe used to amplify corresponding polynucleotides from a chosen organismby PCR. This technique may be used to isolate additional codingsequences from a desired organism or as a diagnostic assay to determinethe presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook et al. (1989),supra.

Similarly, the gene sequences disclosed herein, or fragments thereof,can be used as hybridization probes to screen and find other esteraseproducing organisms.

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with >90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook, supra

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seencbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalentprogram” is intended to mean any sequence comparison program that, forany two sequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, and the like.

In some embodiments, expression cassettes comprising a promoter operablylinked to a heterologous nucleotide sequence of the invention thatencodes an FE polypeptide are further provided. The expression cassettesof the invention find use in generating transformed plants, plant cells,and microorganisms and in practicing the methods for production of FEactivity disclosed herein. The expression cassette will include 5′ and3′ regulatory sequences operably linked to a polynucleotide of theinvention. “Operably linked” is intended to mean a functional linkagebetween two or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites and/or recombination sites for insertion of the polynucleotidethat encodes an FE polypeptide to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter),translational initiation region, a polynucleotide of the invention, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theinvention may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theinvention may be heterologous to the host cell or to each other. As usedherein, “heterologous” in reference to a sequence is a sequence thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked polynucleotide of interest, may be native with the plant host, ormay be derived from another source (i.e., foreign or heterologous) tothe promoter, the polynucleotide of interest, the host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639. In particular embodiments, the potato proteaseinhibitor II gene (PinII) terminator is used. See, for example, Keil etal. (1986) Nucl. Acids Res. 14:5641-5650; and An et al. (1989) PlantCell 1:115-122, herein incorporated by reference in their entirety.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed organism. For example, the polynucleotidescan be synthesized using plant-preferred codons for improved expression.See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention,including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome. Awide range of plant promoters are discussed in the recent review ofPotenza et al. (2004) In Vitro Cell Dev Biol—Plant 40:1-22, hereinincorporated by reference. For example, the nucleic acids can becombined with constitutive, tissue-preferred, pathogen-inducible, orother promoters for expression in plants. Such constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

In another aspect, the FE would be expressed upon ingestion by aforaging animal. Exemplary promoters for this aspect would includeSoybean Gmhsp 17.5 promoter and the leucine aminopeptidase (LAP)promoter. The GMhsp promoter is from a heat shock protein gene andinitiates expression if the temperature of the environment is increased.In the laboratory, an increase of 15° C. for 2 hours is the preferredheat shock. However, in non-laboratory conditions suitable increases intemperature will occur in silos and in the rumen of animals that haveingested the plants of this invention. The LAP promoter initiates theexpression of the FE gene upon wounding of the plant. Such woundingwould occur after cutting the plant or after mastication by a foraginganimal.

Tissue-preferred expression may be accomplished by tissue-preferredpromoters, such as leaf-preferred, vascular tissue-preferred, orstalk-preferred promoters. Similarly, the timing of FE expression may beof significant importance, and accordingly, temporal promoters may bedesired. For example, if expression of FE is desired after the plant hasbeen cut, removed from the ground or ingested, an appropriate promoterwould be a senescence promoter. For example, the promoter of BFN1 couldbe used, since BFN1 has recently been shown to be a nuclease expressedin senescing leaves, Perez-Amador, et al., (2000) Plant Physiol.122:169. Similarly, the promoter of SAG12, a cysteine protease which isalso found in senescing leaves, could be used (Noh & Amasino, (1999)Plant Mol. Biol. 41:181). Moreover, the polypeptides of the inventionmay also be targeted to specific subcellular locations within a plantcell.

Additionally, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

In certain embodiments the nucleic acid sequences of the embodiments canbe stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired phenotype. This stacking may beaccomplished by a combination of genes within the DNA construct, or bycrossing Rcg1 with another line that comprises the combination. Forexample, the polynucleotides of the embodiments may be stacked with anyother polynucleotides of the embodiments, or with other genes. Thecombinations generated can also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides of the embodimentscan also be stacked with any other gene or combination of genes toproduce plants with a variety of desired trait combinations includingand not limited to traits desirable for animal feed such as high oilgenes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and5,703,409); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122); and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)), thedisclosures of which are herein incorporated by reference. Thepolynucleotides of the embodiments can also be stacked with traitsdesirable for insect, disease or herbicide resistance (e.g., Bacillusthuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;5,737,514; 5723,756; 5,593,881; Geiser et al (1986) Gene 48:109);lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS genes, GAT genes such as those disclosed in U.S.Patent Application Publication US2004/0082770, also WO02/36782 andWO03/092360)); and traits desirable for processing or process productssuch as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g.,fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516));modified starches (e.g., ADPG pyrophosphorylases (AGPase), starchsynthases (SS), starch branching enzymes (SBE) and starch debranchingenzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No.5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)),the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the embodiments with polynucleotidesproviding agronomic traits such as male sterility (e.g., see U.S. Pat.No. 5,583,210), stalk strength, flowering time, or transformationtechnology traits such as cell cycle regulation or gene targeting (e.g.WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which areherein incorporated by reference.

These stacked combinations can be created by any method including andnot limited to cross breeding plants by any conventional or TopCross®methodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide. In some embodiments, thepolynucleotide will be presented in such a manner that the sequencegains access to the interior of a cell of the plant, including itspotential insertion into the genome of a plant. The methods of theinvention do not depend on a particular method for introducing asequence into a plant, only that the polynucleotide gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotides into plants are known in the art including, but notlimited to, stable transformation methods, transient transformationmethods, and virus-mediated methods. Polypeptides can also be introducedto a plant in such a manner that they gain access to the interior of theplant cell or remain external to the cell but in close contact with it.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” or “transient expression” is intended to meanthat a polynucleotide is introduced into the plant and does notintegrate into the genome of the plant or a polypeptide is introducedinto a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include, but are not limited to,microinjection (Crossway et al. (1986) Biotechniques 4:320-334),electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055- and 5,981,840), direct gene transfer (Paszkowski et al.(1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see,for example, Sanford et al., U.S. Pat. Nos. 4,945,050; 5,879,918;5,886,244; and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, andOrgan Culture Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058). Also see, Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the FE sequences of the invention can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the FE protein or variants and fragments thereofdirectly into the plant or the introduction of the FE protein transcriptinto the plant. Such methods include, for example, microinjection orparticle bombardment. See, for example, Crossway et al. (1986) Mol. Gen.Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler etal. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994)The Journal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, the polynucleotide can betransiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it's released to become integrated into thegenome is greatly reduced. Such methods include the use particles coatedwith polyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. It is recognized that the an FE polypeptide of the inventionmay be initially synthesized as part of a viral polyprotein, which latermay be processed by proteolysis in vivo or in vitro to produce thedesired recombinant protein. Further, it is recognized that promoters ofthe invention also encompass promoters utilized for transcription byviral RNA polymerases. Methods for introducing polynucleotides intoplants and expressing a protein encoded therein, involving viral DNA orRNA molecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant that has stablyincorporated into its genome a target site which is flanked by twonon-recombinogenic recombination sites that correspond to the sites ofthe transfer cassette. An appropriate recombinase is provided and thetransfer cassette is integrated at the target site. The polynucleotideof interest is thereby integrated at a specific chromosomal position inthe plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having expression of the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Pedigree breeding starts with the crossing of two genotypes, such as anelite line of interest and one other elite inbred line having one ormore desirable characteristics (i.e., having stably incorporated apolynucleotide of the invention, having a modulated activity and/orlevel of the polypeptide of the invention, etc) which complements theelite line of interest. If the two original parents do not provide allthe desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneous lines asa result of self-pollination and selection. Typically in the pedigreemethod of breeding, five or more successive filial generations ofselfing and selection is practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed inbred. In specificembodiments, the inbred line comprises homozygous alleles at about 95%or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. As discussed previously, backcrossing can be used totransfer one or more specifically desirable traits from one line, thedonor parent, to an inbred called the recurrent parent, which hasoverall good agronomic characteristics yet lacks that desirable trait ortraits. However, the same procedure can be used to move the progenytoward the genotype of the recurrent parent but at the same time retainmany components of the non-recurrent parent by stopping the backcrossingat an early stage and proceeding with selfing and selection. Forexample, an F1, such as a commercial hybrid, is created. This commercialhybrid may be backcrossed to one of its parent lines to create a BC1 orBC2. Progeny are selfed and selected so that the newly developed inbredhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the non-recurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newhybrids and breeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of maize inbred line of interest, comprising thesteps of crossing a plant of maize inbred line of interest with a donorplant comprising a mutant gene or transgene conferring a desired trait(i.e., increased digestibility), selecting an F1 progeny plantcomprising the mutant gene or transgene conferring the desired trait,and backcrossing the selected F1 progeny plant to the plant of maizeinbred line of interest. This method may further comprise the step ofobtaining a molecular marker profile of maize inbred line of interestand using the molecular marker profile to select for a progeny plantwith the desired trait and the molecular marker profile of the inbredline of interest. In the same manner, this method may be used to producean F1 hybrid seed by adding a final step of crossing the desired traitconversion of maize inbred line of interest with a different maize plantto make F1 hybrid maize seed comprising a mutant gene or transgeneconferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topcrossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development” by Fehr, (1993)Macmillan Publishing Company, the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plant can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

As mentioned above, the transgenic plants of this invention can be usedas feed or for silage production for animals, such as cattle, sheep,goats, horses, pigs, poultry, and other livestock. In addition, themethods of this invention can be used to transform any plant into whichFE expression is desired. For example, it is advantageous to break downcell walls during biomass conversion or during processing of plants forfoodstuffs. This invention would help to achieve this goal moreeffectively and inexpensively.

The inventive methods herein may also be used to provide enzymes toenhance the availability of fermentable sugars in plants, throughpolypeptide addition to plant material or via fermentation withrecombinant bacteria containing the polynucleotides. Carbohydrates maybe subject to further modification, either exogenously or endogenously,by the action of other enzymes. Such enzymes include, but are notlimited to, endoglucanases, xylosidases and/or cell biohydrolases. Theseenzymes may be provided either in an expression cassette provided forherein (i.e., endogenous) or applied to the plant cell walls (i.e.,exogenous) to enhance the availability of mono- and/or di-saccharides.

Plants other than grasses may find a use in the present invention. Forexample, corn (or maize) is contemplated to be useful. The grass Festucais similar to maize in cell wall structure and therefore provides a goodmodel of the ability to enhance the availability of fermentablecarbohydrates in corn. Other useful plants contemplated for use in thepresent invention include, but are not limited to, Festuca, Lolium, Zea,Avena, Sorghum, Millet (tropical cereals), Miscanthus (a grass withpotential for use as a biomass energy crop), Cenchrus, Dichanthium,Brachiaria and Paspalum (apomictic tropical range grasses) and Poa(Kentucky bluegrass).

A gene encoding an FE polypeptide of the invention may be introducedinto any suitable microbial host according to standard methods in theart. For example, microorganism hosts that are known to occupy the“phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplana) of one or more crops of interest may be selected. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, and to provide for stable maintenance and expression ofthe gene expressing the protein.

Genes encoding FE polypeptides of the invention, or fragments thereof,can also be used to transform bacteria with the intention of using thetransformed bacteria as a silage inoculant, probiotic, food starterculture or for use in the production of food or feed additives byfermentation. Organisms that would be useful in such situations include,but are not limited to, Lactobacillus, Lactococcus, Enterococcus,Pediococcus and Leuconostoc. Likewise, the genes encoding the FEpolypeptides of the invention, or fragments thereof, can be used totransform bacteria for the purposes of biofuel production (See Tabka, M.G. et al. (2006) Enzyme and Microbial Technology 39: 897-902.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Pediococcus, Enterococcus, Rhodopseudomonas, Methylius, Agrobacterium,Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, andAlcaligenes, fungi, particularly yeast, e.g., Saccharomyces,Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, andAureobasidium. Other organisms of interest are such phytospherebacterial species as Pseudomonas syringae, Pseudomonas fluorescens,Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, Clavibacter xyli and Azotobacter vinlandir and phytosphereyeast species such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesrosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Also of interest are the pigmented microorganisms.

Other illustrative prokaryotes, both Gram-negative and gram-positive,include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium;Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas,Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae,such as Pseudomonas and Acetobacter; Azotobacteraceae andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Microbial host organisms of particular interest include yeast, such asRhodotorula spp., Aureobasidium spp., Saccharomyces spp., andSporobolomyces spp., phylloplane organisms such as Pseudomonas spp.,Erwinia spp., and Flavobacterium spp., and other such organisms,including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more elements.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

The following examples are provided by way of illustration, not by wayof limitation.

EXPERIMENTAL Example 1 Ferulate Esterase Gene Cloning

Total DNA from Lactobacillus buchneri strain LN4017, deposited as PatentDeposit No. PTA-6138, was used to generate an E. coli clone whichexpressed FE activity. Size-fractionated partial Sau3AI digested totalDNA libraries of LN4017 were constructed. Approximately 1000 clones werescreened for FE activity in E. coli DH5alpha by looking for zones ofclearing on Difco™ Luria-Bertani agar plates containing 50 μg/mLAmpicillin, 80 μM IPTG and 0.12% ethyl 4-hydroxy-3-methoxycinnamate. OneFE positive clone was identified and found to contain a 3 kb insert inpUC18. The plasmid DNA was transformed into E. coli Novablue cells and anumber of transformants were generated including clone EF-1. This clonealso showed zones of clearing on agar plates.

The 3 kb EF-1 clone was cut into smaller fragments with convenientrestriction enzymes and sub-cloned into pUC18, using DH5alpha as thecloning host. Removal of a 0.45 kb fragment from one end generatedsub-clone A which retained activity on agar plates. The remaining twofragments were used to generate sub-clone B, which was 1.4 kb in lengthand sub-clone C, which was 1.2 kb in length (see FIG. 3). Backgroundesterase activity (or possibly pH drop) from the E. coli host made itdifficult to determine whether sub-clones B and C had activity on agarplates.

Example 2 Ferulate Esterase Gene Sequence Analysis

DNA sequencing was conducted on clone EF-1 and on sub-clones A, B and C.Sequence analysis of an ORF (SEQ ID NO: 2) from sub-clone B indicatedthat it encoded a peptide (SEQ ID NO: 3) of 260 amino acids with a 28kDa molecular weight. BLASTX analysis of SEQ ID NO: 2 detected a numberof amino acid homologies including homology to alpha/beta hydrolases(including Accession Nos: AAK81587, AAM25001), Lactobacillus esterases(including Accession Nos: AAV43644, CAD65143) and cinnamoyl esterhydrolases (including Accession No: ZP_(—)00732443). Esterase activesite residues appeared to be conserved.

Sub-clone C was resistant to sequencing on one end, possibly due tosecondary structure within the DNA. Multiple primers from bothdirections were attempted. The region was ultimately sequenced using DNAfrom the 3 kb clone EF-1 and sub-clone A and the resulting ORF is setforth herein as SEQ ID NO: 4, which encodes SEQ ID NO: 5. BlastXanalysis of SEQ ID NO: 4 generated hits to acetyl esterases, includingone from L. plantarum (Accession No: CAD64912). There was some homologydetected to a Neurospora crassa feruloyl esterase B precursor (AccessionNo: CAC05587).

The peptide encoded by ORF1 is set forth in SEQ ID NOs: 3 and has beenshown to be a member of the alpha/beta hydrolase-fold family of enzymes,which contain a catalytic triad of amino acids consisting ofnucleophile-acid-histidine. These three amino acids are separated by avariable number of amino acid residues in the primary sequence, but arelocated in similar topological locations in the native protein. In mostcases, the nucleophile is a serine residue. (Diaz, E., Timmis, K. N.(1995) The Journal of Biological Chemistry Vol. 270 No. 11, 6403-6411).GAP alignments of the peptide of SEQ ID NO: 3 with other hydrolasesdemonstrated conservation of the catalytic triad (Ser, Asp and His).(See FIGS. 1 and 2) In addition, the peptide showed the conservedpentapeptide sometimes referred to as the nucleophile elbow. Thispentapeptide motif is characterized by a glycine followed by any aminoacid, followed by a serine, followed by any amino acid, followed byanother glycine (Schrag, J. D. and Cygler, M. (1997) Methods inEnzymology Vol 284: 85-107).

Sub-clone C does not appear to contain the complete open reading frame;the first few amino acids appear to be encoded by the end of sub-clone B(see FIG. 3). The putative ORF of 266 amino acids encodes a 28 kDaprotein.

Example 3 Ferulate Esterase and Acetyl Esterase Activity Assays

The FE and AE activities of the 3 kb clone, EF-1 (discussed in Example1), and of sub-clones A, B and C, were assayed according to theprotocols described below. As shown in Table 1, cell lysates from cloneEF-1 (which contains the ORFS for the peptides of SEQ ID NOs: 3 and 5)and sub-clone A (which is missing an upstream non-coding region) hadstrong FE and AE activity. Sub-clone B (which contains the ORF for thepeptide of SEQ ID NO: 3) had FE activity but lacked AE activity,relative to the vector-only control. Sub-clone C (which encodes asmaller portion of the peptide of SEQ ID NO: 5) had neither AE activitynor FE activity, relative to the vector-only control. This data suggeststhat the peptide of SEQ ID NO: 3 is an FE enzyme, while the peptide ofSEQ ID NO: 5 is an AE enzyme.

TABLE 1 Activity Levels of Cleared Lysates. Activity is expressed asnmoles pNP released/min/mg protein. Sample Details¹ FE² AE³ pUC18 vectoronly 0.11 0.72 Clone EF-1 entire insert 3.39 3.70 subclone A Subclone -missing part of 3.13 3.38 upstream non-coding region subclone BSubclone - contains Est1 1.05 0.72 subclone C Subclone - contains partof Est2 0.11 0.60 ¹ E. coli host strain DH5alpha was used for allrecombinants ²FE, Ferulic Acid Esterase ³AE, Acetyl Esterase

Determination of Acetyl or Ferulate Esterase Activity

Lactic acid bacterial cultures were grown in De Man Rogosa Sharpe broth(MRS broth; Difco™ Lactobacilli MRS; Becton Dickinson and Company,Sparks, Md. 21152 USA), prepared as described by the manufacturer, for24 to 48 hours. E. coli cultures were grown with aeration in Difco™Luria-Bertani broth containing 50 μg/mL ampicillin for 16-17 hours. Thebacterial cells were harvested by centrifugation (18000×g; 5 min) andresuspended in PBS, Phosphate Buffered Saline, containing sodium azide(10 μg/mL). Cells were lysed using a French Press (Thermo SpectronicInstruments, Inc., Rochester, N.Y.) as is known in the art. Clearedlysates were obtained by centrifugation at 18000×g for 10 min. Testsamples (cleared lysates or soluble protein) were then assayed foracetyl or ferulate esterase activity using methods described previously(Huggins and Lapides (1947) J. Biol. Chem. 170: 467-482; Mastihuba etal. (2002) Analytical Biochemistry 309, 96-101) with modifications asdetailed below.

The substrates for AE activity, 4-nitrophenyl acetate (4NPAc,Sigma-Aldrich, St. Louis, Mo.); and FE activity, 4-nitrophenyl ferulate(4NPF, Institute of Chemistry, Slovak Academy of Sciences Dubrayska,Cesta 9, 845 38, Slovakia) were dissolved in dimethyl sulphoxide (DMSO)and diluted 10-fold to the final working substrate solution of 2.5 mM in0.5 M potassium phosphate containing 2.5% Triton X-100 (pH6.0 for 4NPAc,pH7.0 for 4NPF). Using microtiter plates, reaction mixtures contained 80μL of substrate and 20 μL of test sample and were incubated at 37° C.for 60 min. Control wells consisting of 1) 4NPAc or 4NPF substratesolution and 2) test samples were included and otherwise treated thesame way as the reaction mixtures. Following the incubation period, eachreaction was diluted 10-fold in 0.5 M potassium phosphate containing 10%DMSO (pH 8.0) and the optical density was determined at 405 nm(SpectraMax 190 Microplate Reader, Molecular Devices, Menlo ParkCalif.). Reaction mixture absorbance readings were corrected forabsorbance readings of controls. Concentration of p-nitrophenol wasdetermined by reference to a standard curve. Protein concentrations weredetermined according to the method published by Bradford (Bradford, M,(1976) Analytical Biochemistry 72 248-254). AE or FE activities of thesamples were expressed as nmoles of p-nitrophenol (pNP) released perminute per mg of protein.

Example 4 Ferulate Esterase Gene Protein Expression

In order to express the LN4017 esterase genes in E. coli, the ORF setforth in SEQ ID NO: 2 and 4 were PCR cloned in the Invitrogen Gateway®cloning vector pDONR221. Two Gateway® clones, pENTR221-Est1 andpENTR221-Est 2, recombined into the Gateway® vector pET28-His 7. The Est1 and Est 2 proteins (SEQ ID NO: 3 and 5, respectively) were expressedwith histidine fusion tags in E. coli strain BL21 (DE3) Gold andpurified using standard protocols for column elutions. Soluble proteinelution fractions were used to run enzyme assays as described in Example3 and were compared to cleared cell lysate from L. buchneri LN4017. Asshown in Table 2, although both elution proteins display both FE and AEactivity, the Est 1 protein displays a much stronger FE activity, whileEst 2 displays stronger AE activity. This is consistent with the resultsof Example 3 and the Blast X analysis of the ORFs. The weaker activitieswere not detected until the ORFs were placed into the T7 expressionvector.

TABLE 2 FE and AE Activity of Purified Proteins. Activity is expressedas nmoles pNP released/min/mg protein Sample FE¹ AE² LN4017 10.47 14.09HisT7-Est1 (SEQ ID NO: 3) 142.04 13.14 HisT7-Est2 (SEQ ID NO: 5) 43.7066.72 ¹FE, Ferulic Acid Esterase ²AE, Acetyl Esterase

Example 5 Transformation and Regeneration of Transgenic Maize Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing a nucleotide sequence encoding the FE polypeptide setforth in SEQ ID NO:1 operably linked to a promoter that drivesexpression in a maize plant cell and a selectable marker (e.g., theselectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37),which confers resistance to the herbicide Bialaphos). Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising a nucleotide sequence encoding the FEpolypeptide set forth in SEQ ID NO:3 operably linked to a promoter thatdrives expression in a maize cell is made. This plasmid DNA plus plasmidDNA containing a selectable marker (e.g., PAT) is precipitated onto 1.1μM (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

-   -   100 μL prepared tungsten particles in water    -   10 μL (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)    -   100 μL 2.5 M CaCl₂    -   10 μL 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multi-tube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/L Bialaphos,and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for fungal resistance.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/L Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/L silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/L N6 basalsalts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D (brought tovolume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/LGelrite (added after bringing to volume with D-I H₂O); and 0.85 mg/Lsilver nitrate and 3.0 mg/L bialaphos (both added after sterilizing themedium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/L glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/L Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/L indoleacetic acidand 3.0 mg/L bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinicacid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/Lglycine brought to volume with polished D-I H₂O), 0.1 g/L myo-inositol,and 40.0 g/L sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/L bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 6 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with a nucleotidesequence encoding the polypeptide of SEQ ID NO:3, the method of Zhao isemployed (U.S. Pat. No. 5,981,840, and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium, where the bacteria arecapable of transferring the polynucleotide construct to at least onecell of at least one of the immature embryos (step 1: the infectionstep). In this step the immature embryos are immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos are cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is performed. In this resting step, the embryosare incubated in the presence of at least one antibiotic known toinhibit the growth of Agrobacterium without the addition of a selectiveagent for plant transformants (step 3: resting step). The immatureembryos are cultured on solid medium with antibiotic, but without aselecting agent, for elimination of Agrobacterium and for a restingphase for the infected cells. Next, inoculated embryos are cultured onmedium containing a selective agent and growing transformed callus isrecovered (step 4: the selection step). The immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A DNA construct comprising a promoter that drives expression in aplant or plant cell operably linked to a nucleotide sequence thatencodes a polypeptide having ferulate esterase activity, wherein thenucleotide sequence is selected from the group consisting of: a) apolynucleotide comprising SEQ ID NO: 1 or 4; b) a polynucleotide havingat least about 85% sequence identity to SEQ ID NO: 1 or 4; c) apolynucleotide encoding SEQ ID NO: 5; and d) a polynucleotide encoding apolypeptide having at least 85% sequence identity to SEQ ID NO:
 5. 2. Atransformed plant cell comprising at least one DNA construct accordingto claim
 1. 3. The plant cell of claim 2, wherein said plant cell isfrom a monocot.
 4. The plant cell of claim 3, wherein said monocot is amember of a genus selected from the group consisting of Festuca, Lolium,Sorghum, Zea, Triticum, Avena, and Poa.
 5. A transgenic plant comprisingat least one DNA construct according to claim
 1. 6. The transgenic plantof claim 5, wherein said plant displays increased digestibility.
 7. Thetransgenic plant of claim 6, wherein said plant is a monocot.
 8. Thetransgenic plant of claim 7, wherein said monocot is a member of a genusselected from the group consisting of Festuca, Lolium, Sorghum, Zea,Triticum, Avena, and Poa.
 9. A transformed seed of the transgenic plantof claim 5, wherein the seed comprises the construct.
 10. The transgenicplant of claim 5, further comprising introduction into the plant asecond DNA construct comprising a promoter operably linked to a xylanaseencoding polynucleotide.
 11. A method for increasing digestibility of aplant or plant part fed to an animal, the method comprising introducinginto a plant a DNA construct comprising a promoter operably linked to apolynucleotide encoding ferulate esterase, wherein said polynucleotidehas a sequence selected from the group consisting of: a) apolynucleotide comprising SEQ ID NO: 1 or 4; b) a polynucleotide havingat least about 85% sequence identity to SEQ ID NO: 1 or 4; c) apolynucleotide encoding SEQ ID NO: 5; and d) a polynucleotide encoding apolypeptide having at least 85% sequence identity to SEQ ID NO:
 5. 12.The method of claim 11, wherein said plant is a monocot.
 13. The methodof claim 12, wherein said monocot is a member of a genus selected fromthe group consisting of Festuca, Lolium, Sorghum, Zea, Triticum, Avena,and Poa.
 14. The method of claim 11, further comprising introductioninto the plant a second DNA construct comprising a promoter operablylinked to a xylanase encoding polynucleotide.
 15. An isolated nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of: a) a polynucleotide comprising the sequence set forth inSEQ ID NOs: 1 or 4; b) a polynucleotide having at least about 85%sequence identity to SEQ ID NOs: 1 or 4; c) a polynucleotide encodingthe amino acid sequence of SEQ ID NO: 5; and d) a polynucleotideencoding the amino acid sequence of a polypeptide having at least 85%sequence identity to SEQ ID NOs:5, wherein said polypeptide has ferulateesterase activity.
 16. The nucleic acid molecule of claim 15, whereinsaid nucleotide sequence is optimized for expression in a plant.
 17. ADNA construct that expresses in a bacterial cell, comprising apolynucleotide sequence that encodes a polypeptide having ferulateesterase activity, wherein the nucleotide sequence is selected from thegroup consisting of: a) a polynucleotide comprising SEQ ID NO: 1, 2 or4; b) a polynucleotide having at least about 85% sequence identity toSEQ ID NO: 1, 2 or 4; c) a polynucleotide encoding SEQ ID NO: 3 or 5;and d) a polynucleotide encoding a polypeptide having at least 85%sequence identity to SEQ ID NO: 3 or
 5. 18. A transformed bacterial cellcomprising at least one DNA construct according to claim
 17. 19. Thetransformed bacterial cell of claim 18, wherein the bacteria is from aLactobacillus species.
 20. The transformed bacterial cell of claim 18,where the bacteria is used as a silage incoculant.